High stringency blot hybridization analysis of restricted cellular DNA from the different species of mice, using cloned MMTV proviral DNA as a probe, has led to the identification of M. musculus, M. caroli, and M. pahari breeding colonies which lack the genetically transmitted MMTV genome (designated MMTV-negative mice). One other M. musculus colony was found to contain only the MMTV long terminal repeat region of the viral genome in its germline. By lowering the stringency of blot hybridization, we have identified a new class of highly diverged MMTV-related sequences (designated MMTV-B) in the cellular DNA of all species of the genus Mus, including MMTV-negative mice. Sequences related to the MMTV genome have also been detected in human cellular DNA using low stringency blot hybridization conditions. Several recombinant clones of human cellular DNA containing MMTV-related sequences have been isolated. The major region of homology corresponds to the gag-pol genes of the MMTV genome.